Scanning microscope

ABSTRACT

The scanning microscope includes an illumination beam path, microscope optics and at least one light source which generates an excitation light beam of a first wavelength and a second light beam of a second wavelength. Microscope optics are provided for focussing the excitation light beam onto a first focal region in a first plane of a sample and for focusing the second light beam onto a second focal region in a second plane of the sample. The first focal region and the second focal region overlap partially. The optical properties of the components arranged in the illumination beam path are matched to one another such that optical aberrations are corrected in such a way that the focal regions remain static relative to one another irrespective of the scanning movement.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 10/023,187, filed Dec.17, 2001 now U.S. Pat. No. 6,914,236, which application claims priorityto German patent application 100 63 276.9-42, filed Dec. 19, 2000. Theentire subject matter of both of these prior applications is herebyincorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to a scanning microscope having an illuminationbeam path, microscope optics and having at least one light source, whichgenerates an excitation light beam of a first wavelength and an emissionlight beam of a second wavelength, the excitation light beam beingfocussed onto a first focal region in a first plane and the emissionlight beam being focussed onto a second focal region in a second planein a sample.

BACKGROUND OF THE INVENTION

In scanning microscopy, a sample is illuminated with a light beam inorder to observe the reflected or fluorescent light emitted by thesample. The focus of the illumination light beam is moved in an objectplane with the aid of a controllable beam-deflection device, generallyby tilting two mirrors, the deflection axes usually being mutuallyperpendicular so that one mirror deflects in the x direction and theother deflects in the y direction. The mirrors are tilted, for example,with the aid of galvanometer control elements. The power of the lightcoming from the object is measured as a function of the position of thescanning beam. The control elements are usually equipped with sensors toascertain the current mirror setting.

Especially in confocal scanning microscopy, an object is scanned withthe focus of a light beam in three dimensions.

A confocal scanning microscope generally comprises a light source,focusing optics by which the light from the light source is focused ontoa pinhole (the so-called excitation aperture), a beam splitter, abeam-deflection device for beam control, microscope optics, a detectionaperture and the detectors for registering the detection or fluorescentlight. The illumination light is usually input via a beam splitter. Thefluorescent or reflected light coming from the object travels back viathe beam-deflection device to the beam splitter, and passes through thelatter in order to be subsequently focused onto the detection aperture,behind which the detectors are located. Detection light which does notoriginate directly from the focus region takes a different light pathand does not pass through the detection aperture, so that pointinformation is obtained which leads to a three-dimensional image bysequential scanning of the object. A three-dimensional image is usuallyachieved through layer-by-layer imaging. Instead of guiding illuminationlight over or through the object using a beam-deflection device, it isalso possible to move the object while the illumination light beam isstatic. Both scanning methods, beam scanning and object scanning, areknown and widespread.

The power of the light coming from the object is measured at set timeintervals during the scanning process, and hence scanned scan-point byscan-point. The measurement value must be assigned uniquely to therelevant scan position, so that an image can be generated from themeasurement data. To that end, it is expedient to measure the state dataof the adjustment elements of the beam-deflection device continuously atthe same time or, although this is less accurate, to use directly thesetpoint control data of the beam-deflection device.

It is also possible in a transmitted-light arrangement, for example, todetect the fluorescent light or the transmission of the excitation lighton the condenser side. The detection light beam does not then travel tothe detector via the scanning mirrors (non-descan arrangement). Fordetection of the fluorescent light, the transmitted-light arrangementwould need a detection aperture on the condenser side in order toachieve three-dimensional resolution, as in the described descanarrangement. In the case of two-photon excitation, however, a detectionaperture on the condenser side can be omitted since the excitationprobability depends on the square of the photon density (˜intensity²),which is naturally much higher at the focus than in the neighbouringregions. The vast majority of the fluorescent light to be detectedtherefore originates with high probability from the focus region, whichobviates the need for further differentiation, using an aperturearrangement, between fluorescence photons from the focus region andfluorescence photons from the neighbouring regions.

The resolving power of a confocal scanning microscope is dictated, interalia, by the intensity distribution and the spatial extent of the focalregion of the illumination light beam. An arrangement to increase theresolving power for fluorescence applications is known fromPCT/DE/95/00124. This arrangement comprises a light source, whichgenerates an excitation light beam of a first wavelength and an emissionlight beam of a second wavelength, the excitation light beam beingfocussed onto a first focal region and the emission light beam beingfocussed onto a second focal region in a sample, which overlapspartially with the first focal region. The excitation light beam excitesoptically the sample in the first focal region, while the emission lightbeam generates stimulated emission in the second focal region. Only thespontaneously emitted light from the part of the first focal region inwhich no stimulated emission has been generated is then detected, sothat an improvement in the resolution is achieved overall. The term STED(Stimulated Emission Depletion) has become attributed to this method.

STED technology has been developed further to the extent that anincrease in the resolution can be achieved both laterally and axially,by providing the focal region of the emission light beam with anintensity distribution which vanishes on the inside. Expressed simply,the focal region is, so to speak, internally hollow. Such an intensitydistribution can be achieved, for example, with the aid of a λ/2 plate,which is fitted in a Fourier plane relative to the focal plane of theemission light beam, whose diameter is less than the beam diameter andwhich is consequently illuminated all round. The focal region of theemission light beam must be made congruent with the focal region of theexcitation light beam. Only spontaneously emitted light from the regionof vanishing intensity in the focal region of the emission light beamwill then still be detected. In theory, resolutions far smaller than 100nm can be achieved with such arrangements.

It is important that the focal regions of the emission light beam andthe excitation light beam be made to overlap suitably.

Even well-corrected high-end optical elements have residual aberrations,which are usually negligible in conventional microscopy but becomehighly significant in the resolution range considered here. Inparticular, owing to residual chromatic aberrations, the differingwavelengths of the emission light beam and the excitation light beamlead to serious errors. For example, just the axial chromatic aberrationof high-end microscope objectives amounts to about 150 nm, and istherefore above the resolving power theoretically achievable with STED.In the case of a beam-scanning system, lateral aberrations are alsoadded to the axial aberrations, so that the overlap region varies bothaxially and laterally during the scanning movement.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a scanningmicroscope having optical means which are configured in such a way thata resolution required for STED microscopy is achievable.

The present invention provides a scanning microscope including:

-   -   at least one light source for generating an excitation light        beam of a first wavelength and an emission light beam of a        second wavelength,    -   microscope optics for focusing the excitation light beam onto a        first focal region in a first plane of a sample and for focusing        the emission light beam onto a second focal region in a second        plane of the sample, whereby the excitation light beam optically        excites the sample in the first focal region and the emission        light beam generates stimulated emission in the second focal        region, and whereby the first and second focal regions are        overlapping at least partially,    -   said light source and said microscope optics defining an        illumination beam path    -   means for scanning the excitation light beam and the emission        light beam onto a sample,    -   components for guiding and shaping being arranged in the        illumination beam path, whereby optical properties of the        components and of the microscope optics are matched to one        another such that optical aberrations are corrected in such a        way that the focal regions remain static relative to one another        irrespective of the scanning movement.

The invention has the advantage that the theoretical resolving power canbe achieved in both object-scanning and beam-scanning systems.

It is important that the focal regions of the emission light beam andthe excitation light beam be made to overlap suitably. Furthermore, thisoverlap must also be preserved when scanning the sample. Overlappinginvolves a spatial interrelationship of the two light beams, which willnot be changed by the scanning process.

According to the invention, in particular, chromatic aberrations such asaxial chromatic aberration, chromatic difference of magnification orlateral chromatic aberration, are corrected. Such correction can beachieved in a particularly advantageous way by extra optics in thesubsidiary beam paths, of the illumination-light beam path, along whichonly the excitation light beam or only the emission light beam travels.In these subsidiary beam paths, the axial and lateral beam propertiescan be specifically influenced. It is then possible to compensate forany remaining axial chromatic aberration, for example, by providingoptical paths of different lengths between the focal regions and thelight sources of the excitation light beam and the emission light beam.

It is also advantageous to correct monochromatic aberrations such asspherical aberrations, coma, astigmatism, field curvature or distortion,by extra optics in the subsidiary beam paths, of the illumination-lightbeam path, along which only the excitation light beam or only theemission light beam travel. Nevertheless, correction in the part of theillumination-light beam path along which the excitation light beam andthe emission light beam travel together is also favourable. Thecorrection may involve lenses, drift sections, and also adaptive opticsor active optics. For instance, it is conceivable to use a deformablemirror, for example a sheet mirror or an array of micromirrors, thecurvature or setting of which varies during the scanning movement. AnLCD element, preferably in a Fourier plane relative to the focal plane,which varies the phase of the excitation light beam or the emissionlight beam, or parts of the excitation light beam or the emission lightbeam, may also be provided as adaptive optics in the illumination beampath.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject-matter of the invention is schematically represented in thedrawings and will be described below with the aid of the figures, inwhich:

FIG. 1 shows a schematic representation of the tracks of the focalregions of the excitation light beam and the emission light beam in aconventional system,

FIG. 2 shows a scanning microscope according to the invention, and

FIG. 3 shows a scanning microscope according to the invention in anon-descan arrangement and with multiphoton excitation.

DETAILED DESCRIPTION OF THE INVENTION

FIG. 1 schematically shows the profile of the tracks of the focalregions of the excitation light beam 1 and the emission light beam 3 ina conventional beam-scanning system. The excitation light beam 1 and theemission light beam 3 are focussed by the microscope optics 5. The focalregion 7 of the excitation light beam is represented by solid lines. Itfollows the line 11 as the scanning movement is executed. The focalregion 9 of the emission light beam is represented by dashes. It followsthe line 13 as the scanning movement is executed. The overlap region 15changes as the scanning movement is executed. Owing to axial chromaticaberration, the focal regions 7 and 9 do not become congruent even inthe vicinity of the optical axis. Away from the optical axis, this axialaberration is supplemented by the transverse chromatic aberration,together with field curvature or distortion, so that the focal regions 7and 9 are offset both laterally and axially relative to one another.

FIG. 2 shows a scanning microscope according to the invention, which isembodied as a confocal scanning microscope. The first light source 17,which is embodied as a pulse laser, generates the excitation light beam19. The second light source 21, which is also a pulse laser, generatesthe emission light beam 23. The excitation light beam 19 and theemission light beam are combined by the dichroic beam combiner 25 andtravel via the dichroic beam splitter 27 to the scanning module 29,which involves a cardan-suspended scanning mirror 31 that guides theexcitation light beam 19 and the emission light beam 23 via the scanningoptics 33, the optics 35 and, through the microscope optics 37, over orthrough the sample 39. The sample 39 is arranged on a microscope stage(not shown), which permits scanning in the z direction, in the directionof the excitation light beam 19. The various focal planes of the sample39 are scanned successively by the excitation light beam 19 and theemission light beam 23. The excitation light beam 19 and the emissionlight beam 23 form the illumination-light beam path 41, which isrepresented as an unbroken line. The light 43 leaving the sample travelsthrough the microscope optics 37 and, via the scanning module 29, to thebeam splitter 27, passes through the latter and strikes the detector 45,which is embodied as a photomultiplier. The light 43 leaving the sample39 is represented as a dashed line. Electrical detection signalsproportional to the power of the light 43 leaving the object aregenerated in the detector 45 and are sent on to a processing unit (notshown). A bandpass filter 49, which stops out the light with thewavelength of the emission light beam 23, is arranged in front of thedetector. The illumination pinhole 51, which is customarily provided ina confocal scanning microscope, and the detection pinhole 47 areschematically indicated for the sake of completeness. However, some ofthe optical elements for guiding and shaping the light beams are omittedfor the sake of clarity. They are adequately known to a specialistworking in this field. So that the focal regions of the excitation lightbeam 19 and the emission light beam 23 remain static relative to oneanother even while the scanning movement is executed, focussing optics24 are provided between the first light source 17 and the dichroic beamcombiner 25. Together with the different lengths of the optical pathsfrom the first and second light sources 17 and 21 to the dichroic beamcombiner 25, compensation is obtained for the axial chromatic aberrationof all the other optics of the illumination-light beam path 41. Tocompensate for lateral aberrations, adaptive optics 53, which areembodied as an LCD element, are arranged between the second light source21 and the dichroic beam splitter. They are controlled as a function ofthe setting of the scanning mirror 31 in the beam-deflection device 29.

FIG. 3 shows a scanning microscope according to the invention in anon-descan arrangement with multiphoton excitation. In this arrangement,the detection takes place on the condenser side. The illuminationpinhole and the detection pinhole can be omitted in this arrangement.The light 71 leaving the sample 39 is focussed by the condenser optics55 and delivered via the mirror 73 to the detector 49, which is embodiedas a photomultiplier. A filter 75, which stops out the light with thewavelength of the excitation light beam and the emission light beam, isarranged in front of the detector 49. The excitation light beam 63 isgenerated by the first light source 61, which is embodied as aTi:sapphire pulse laser. The emission light beam 69 is generated by thesecond light source 67, which involves an optical parametric oscillator.After combination with the aid of the dichroic beam combiner 59, theillumination of the sample takes place in a similar way to theillumination described in FIG. 2. So that the focal regions of theexcitation light beam 63 and the emission light beam 69 remain staticrelative to one another even while the scanning movement is executed, adefocussing lens 65 is provided between the first light source 61 andthe dichroic beam combiner 59. Together with the different lengths ofthe optical paths from the first and second light sources to thedichroic beam combiner 59, compensation is obtained for the axialchromatic aberration of all the other optics of the illumination-lightbeam path 41. To compensate for lateral aberrations, adaptive optics 57are arranged in the part of the illumination-light beam path 41 whichthe excitation light beam 63 and the emission light beam 69 travelthrough together. They are controlled as a function of the setting ofthe scanning mirror 31 in the beam-deflection device 29.

The invention has been described with reference to a particularembodiment. Of course, however, modifications and amendments may be madewithout thereby departing from the scope of protection of the followingclaims.

PARTS LIST

-   1 excitation light beam-   3 emission light beam-   5 microscope optics-   7 focal region of the excitation light beam-   9 focal region of the emission light beam-   11 line-   13 line-   15 overlap region-   17 first light source-   19 excitation light beam-   21 second light source-   23 emission light beam-   25 dichroic beam combiner-   27 dichroic beam splitter-   29 scanning module-   31 scanning mirror-   33 scanning optics-   35 optics-   37 microscope optics-   39 sample-   41 illumination beam path-   43 emerging light-   45 detector-   47 detection pinhole-   49 bandpass filter-   51 illumination pinhole-   53 adaptive optics-   55 condenser optics-   57 adaptive optics-   59 dichroic beam combiner-   61 first light source-   63 excitation light beam-   65 lens-   67 second light source-   69 emission light beam-   71 emerging light-   73 mirror-   75 filter

1. A scanning microscope comprising: at least one light source forgenerating an excitation light beam of a first wavelength and secondlight beam of a second wavelength, microscope optics for focusing theexcitation light beam onto a first focal region in a first plane of asample and for focusing the second light beam onto a second focal regionin a second plane of the sample, whereby the excitation light beamoptically excites the sample in the first focal region and the secondlight beam optically excites the sample in the second focal region, thefirst and second focal regions overlapping at least partially, saidlight source and said microscope optics defining an illumination beampath, a scanning device for scanning the excitation light beam and thesecond light beam onto the sample, components for guiding and shapingbeing arranged in the illumination beam path, whereby optical propertiesof the components and of the microscope optics are matched to oneanother such that optical aberrations are corrected in such a way thatthe focal regions remain static relative to one another irrespective ofthe scanning movement.
 2. The scanning microscope according to claim 1,whereby the aberrations are chromatic aberrations such as axialchromatic aberration, chromatic difference of magnification or lateralchromatic aberration.
 3. The scanning microscope according to claim 1,whereby the aberrations are monochromatic aberrations such as sphericalaberrations or coma or astigmatism, field curvature or distortion. 4.The scanning microscope according to claim 1 further comprising: anoptical correction device for compensating optical aberrations.
 5. Thescanning microscope according to claim 4, wherein the optical correctiondevice acts only on the excitation light beam.
 6. The scanningmicroscope according to claim 4, wherein the optical correction deviceacts only on the second light beam.
 7. The scanning microscope accordingto claim 4, wherein the optical correction device acts on the excitationlight beam and on the second light beam.
 8. The scanning microscopeaccording to claim 4, wherein the optical correction device includes alens.
 9. The scanning microscope according to claim 4, wherein theoptical correction device includes a drift section.
 10. The scanningmicroscope to claim 4, wherein the optical correction device includesadaptive optics.
 11. The scanning microscope according to claim 10,wherein the adaptive optics includes at least one of an LCD element, amicromirror and a deformable mirror.